cabeza: Biological Overview | References
Gene name - cabeza
Cytological map position - 14B8-14B9
Function - RNA-binding protein
Symbol - caz
FlyBase ID: FBgn0285954
Genetic map position - chrX:16,182,879-16,187,553
Cellular location - potentially cytoplasmic and nuclear
|Recent literature||Lo Piccolo, L. and Yamaguchi, M. (2017). RNAi of arcRNA hsromega affects sub-cellular localization of Drosophila FUS to drive neurodiseases. Exp Neurol 292: 125-134. PubMed ID: 28342748
A conspicuous feature of some neurodegenerative diseases is the loss of nuclear activities of RNA-binding proteins (RBPs) like Fused in sarcoma (FUS) and eventually, their accumulation in cytoplasmic proteinaceous inclusions. A subset of Long non-coding RNAs (lncRNAs) is the core of nuclear bodies (NBs), which are the sites of RNA processing and sequestration of specific ribonucleoproteins (RNPs) complexes. In Drosophila melanogaster the lncRNA hsromega is the architectural RNA (arcRNA) of the NB omega speckles (omega-speckles). This study shows that the neuron-specific and motor neuron-specific knockdown of hsromega impairs locomotion in larval and adult flies and induces anatomical defects in presynaptic terminals of motor neurons, suggesting a novel role of arcRNA hsromega in development of neuromuscular junctions. Since RBPs are recognized as important regulators of neuronal activities, to examine the molecular mechanism of such neurodegeneration, interaction was examined between hsromega and Drosophila orthologue of human FUS (dFUS; Cabeza). Strictly, it was found that dFUS genetically and physically interacts with the arcRNA hsromega. Moreover, a fine regulation of gene expression occurs between hsromega and dFUS and surprisingly, depletion of hsromega was found to affect the sub-cellular compartmentalization of dFUS thus, enhancing its cytoplasmic localization and inducing its loss of nuclear function. The model that is proposed shows the role of arcRNA in diseases affecting the nervous system and in particular it elucidates the molecular mechanism underlying the loss of dFUS nuclear function in the absence of its mutations. These new findings could provide new insights into the pathogenesis of neurodegenerative disease dependent on mis-function or mis-localization of aggregation prone RNA binding proteins like FUS in Amyotrophic Lateral Sclerosis.
|Machamer, J. B., Woolums, B. M., Fuller, G. G. and Lloyd, T. E. (2018). FUS causes synaptic hyperexcitability in Drosophila dendritic arborization neurons. Brain Res [Epub ahead of print]. PubMed ID: 29625118
Mutations in the nuclear localization signal of the RNA binding protein FUS cause both Frontotemporal Dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS). These mutations result in a loss of FUS from the nucleus and the formation of FUS-containing cytoplasmic aggregates in patients. To better understand the role of cytoplasmic FUS mislocalization in the pathogenesis of ALS, this study identified a population of cholinergic neurons in Drosophila that recapitulate these pathologic hallmarks. Expression of mutant FUS or the Drosophila homolog, Cabeza (Caz), in class IV dendritic arborization neurons results in cytoplasmic mislocalization and axonal transport to presynaptic terminals. Interestingly, overexpression of FUS or Caz causes the progressive loss of neuronal projections, reduction of synaptic mitochondria, and the appearance of large calcium transients within the synapse. Additionally, overexpression of mutant but not wild type FUS results in a reduction in presynaptic Synaptotagmin, an integral component of the neurotransmitter release machinery, and mutant Caz specifically disrupts axonal transport and induces hyperexcitability. These results suggest that FUS/Caz overexpression disrupts neuronal function through multiple mechanisms, and that ALS-causing mutations impair the transport of synaptic vesicle proteins and induce hyperexcitability.
Mutations in the fused in sarcoma/translated in liposarcoma gene (FUS/TLS, FUS) have been identified in sporadic and familial forms of amyotrophic lateral sclerosis (see Drosophila as a Model for Human Diseases: Amyotrophic lateral sclerosis). FUS is an RNA-binding protein that is normally localized in the nucleus, but is mislocalized to the cytoplasm in ALS, and comprises cytoplasmic inclusions in ALS-affected areas. However, it is still unknown whether the neurodegeneration that occurs in ALS is caused by the loss of FUS nuclear function, or by the gain of toxic function due to cytoplasmic FUS aggregation. Cabeza (Caz) is a Drosophila orthologue of human FUS. This study generated Drosophila models with Caz knockdown, and investigated their phenotypes. In wild-type Drosophila, Caz was strongly expressed in the central nervous system of larvae and adults. Caz did not colocalize with a presynaptic marker, suggesting that Caz physiologically functions in neuronal cell bodies and/or their axons. Fly models with neuron-specific Caz knockdown exhibited reduced climbing ability in adulthood and anatomical defects in presynaptic terminals of motoneurons in third instar larvae. The results demonstrated that decreased expression of Drosophila Caz is sufficient to cause degeneration of motoneurons and locomotive disability in the absence of abnormal cytoplasmic Caz aggregates, suggesting that the pathogenic mechanism underlying FUS-related ALS should be ascribed more to the loss of physiological FUS functions in the nucleus than to the toxicity of cytoplasmic FUS aggregates. Since the Caz-knockdown Drosophila model presented in this study recapitulates key features of human ALS, it would be a suitable animal model for the screening of genes and chemicals that might modify the pathogenic processes that lead to the degeneration of motoneurons in ALS (Sasayama, 2012).
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that is characterized by degeneration of motor neurons, which leads to progressive muscle weakness and eventually fatal paralysis, typically within 1 to 5 years after disease onset. Frontotemporal lobar degeneration (FTLD) is a clinically diverse dementia syndrome, with phenotypes that include behavioral changes, semantic dementia and progressive non-fluent aphasia. Although these two diseases are clinically distinct and affect different parts of the central nervous system, it has been long thought that these two diseases are related since ALS patients often develop cognitive deficits with frontotemporal features and FTLD patients can present symptoms of motor neuron disease. This hypothesis, which was derived from clinical observations, has been biochemically confirmed by identification of the 43 kDa TAR-DNA-binding protein (TDP-43) as the major aggregating protein in subtypes of both ALS and FTLD (ALS-TDP and FTLD-TDP, respectively). Moreover, over 30 different mutations in the TDP-43 gene (TARDBP) have been identified in various sporadic and familial ALS patients, and subsequently TDP-43 mutations were reported in various FTLD-TDP cases. Shortly after the identification of mutations in TDP-43 in ALS cases, mutations in another gene encoding an RNA-binding protein, FUS (fused in sarcoma; also known as TLS, translocated in liposarcoma), were identified in cases with familial ALS (ALS-FUS). Both dominantly and recessively inherited FUS mutations have been reported in familial ALS, and FUS mutations may be more common than TARDBP mutations in familial ALS. Additional mutations in FUS have recently been identified in sporadic ALS cases and in a subset of FTLD cases (FTLD-FUS). FUS is normally a nuclear protein, but cytoplasmic FUS-immunoreactive inclusions were demonstrated in lower motor neurons of ALS patients harboring FUS mutations. Cytoplasmic aggregation of wild-type FUS was subsequently reported as the prominent disease phenotype in other neurodegenerative diseases such as basophilic inclusion body disease, some types of juvenile ALS, and in the majority of tau- and TDP43-negative FTLD. The identification of these two RNA-binding proteins that aggregate and are sometimes mutated in ALS and FTLD gave rise to the emerging concept that disturbances in RNA regulation may play a major role in the pathogenesis of ALS and FTLD. Moreover, FUS aggregation is also demonstrated in Huntington's disease, spinocerebellar ataxia types 1, 2, and 3, and dentatorubropallidoluysian atrophy. These findings suggest an important role for FUS aggregation in the pathogenesis of neurodegenerative diseases beyond ALS and FTLD (Sasayama, 2012).
FUS is a ubiquitously expressed, 526 amino acid protein that was initially identified as a proto-oncogene, and which causes liposarcoma due to chromosomal translocation. FUS is an RNA-binding protein that is implicated in multiple aspects of RNA metabolism including microRNA processing, RNA splicing, trafficking and translation. FUS shows nuclear and cytoplasmic expression and shuttles between the nucleus and the cytoplasm. In neurons, FUS is localized to the nucleus but it is transported to dendritic spines at excitatory post-synapses in a complex with RNA and other RNA-binding proteins. Similar to TDP-43, FUS comprises a glycine-rich domain (GRD), an RNA-recognition-motif (RRM) domain and a nuclear localization sequence (NLS). ALS/FTLD-associated mutations cluster in the C-terminal region of the FUS protein that contains a non-classical R/H/KX2-5PY NLS motif as well as in the GRD motif that is important for protein-protein interactions and also exists in the C-terminal region of TDP-43. Most pathogenic mutations of the TARDBP gene cluster in this GRD motif. The only known genetic cause for ALS/FTLD with FUS pathology is mutations in the FUS gene itself. The FUS mutations in the NLS-containing C-terminal region lead to redistribution of the FUS protein from the nucleus to the cytoplasm. These findings suggest that the loss of physiological nuclear functions of FUS that involve RNA regulation may contribute to the pathogenesis of ALS/FTLD (Sasayama, 2012).
There is a single homolog for each of human FUS and TDP-43 in Drosophila, named Cabeza (Caz) and TAR DNA-binding protein-43 homolog (TBPH), respectively. The Caz gene is located on the X chromosome, and is a member of an RNA binding proteins that are conserved from fly to man. In situ hybridization and immunohistochemical analyses demonstrated that Caz mRNA and protein are enriched in the brain and CNS during embryogenesis, and the Caz protein was detected in the nuclei of several larval tissues and in imaginal discs (Stolow, 1995). The full-length recombinant Caz protein and its RRM domain are capable of binding RNA in vitro (Stolow, 1995). These findings suggest that Caz is a nuclear RNA binding protein that may play an important role in the regulation of RNA metabolism during fly development. Feiguin (2009) reported that Drosophila lacking TBPH presented deficient locomotive behaviors, reduced life span and anatomical defects at neuromuscular junctions (NMJ), suggesting that a loss of TDP-43 nuclear functions could be a causative factor of the neurodegeneration observed in patients with ALS/FTLD (Sasayama, 2012).
As mentioned above, the loss of the nuclear function of FUS or TDP-43 plays an important role in the pathogenesis of ALS/FTLD. However, aggregation of TDP-43 or FUS may by itself be toxic due to a toxic gain-of-function associated with the formation of cytoplasmic aggregates of those proteins, which would trap vital proteins and/or RNAs and might disturb cellular homeostasis. Thus, it remains unclear whether it is the loss of FUS nuclear function or the gain of toxic function resulting from FUS aggregation that is the mechanism that underlies the primary abnormality that leads to the neurodegeneration that occurs in ALS/FTLD. The existence of both dominantly and recessively inherited FUS mutations in familial ALS has provoked further controversy regarding whether the underlying pathogenic mechanism of ALS/FTLD is due to gain-of-toxic-function or loss-of-nuclear function. This study investigated phenotypes of fly models with knockdown of the Drosophila FUS homologue, Caz gene, to provide supporting evidence for the hypothesis that the pathogenesis of ALS/FTLD may be due more to the loss of physiological FUS functions than to the toxicity of its cytoplasmic aggregates. Neuron-specific knockdown of the Drosophila Caz gene reduced the climbing abilities of adult flies as well as caused anatomical defects, such as a reduced length of synaptic branches, in presynaptic terminals of motoneurons in third instar larvae, suggesting that decreased expression of the Drosophila FUS homologue may be sufficient for development of the degeneration of motoneurons and for the deficient locomotive behavior in this model fly (Sasayama, 2012).
It is shown here that Drosophila Caz is strongly expressed in the central nervous system of larvae and adults. Caz did not colocalize with the presynaptic protein Brp, suggesting that Caz performs its physiological functions in neuronal cell bodies and/or their axons. In order to clarify whether or not disruption of the physiological functions of Caz are critical for the development of neurodegeneration even in the absence of abnormal Caz aggregates, fly models were established in which the Caz gene, which is the Drosophila FUS homologue, was knocked down. Neuron-specific knockdown of Caz did not affect the life span of the Caz-knockdown flies but did reduce the climbing abilities of adult flies, and also caused anatomical defects in presynaptic terminals of motoneurons in third instar larvae. These results suggested that a decrease in Caz expression is sufficient for the development of defects in locomotive abilities and for a decrease in the total length of synaptic branches of motoneurons at the NMJs in this Drosophila model. These data may indicate that the loss of physiological FUS functions in motoneurons would be more fundamental than the formation of cytoplasmic FUS aggregates in the pathogenesis of human FUS-related ALS/FTLD (Sasayama, 2012).
To eliminate the possibility that off-target effects of our RNAi construct that contained inverted repeats might generate the observed phenotypes, two different Caz inverted repeat constructs (UAS-Caz-IR1-167 and UAS-Caz-IR363-399) were used whose target sequences did not overlap with each other. Four transgenic fly strains carrying UAS-Caz-IR1-167 were establised. A fly strain carrying UAS-Caz-IR363-399 was obtained from the Vienna Drosophila RNAi center (VDRC). This fly strain carries an RNAi that is targeted to the region corresponding to residues 363-399 of Drosophila Caz (UAS-Caz-IR363-399), These transgenic flies were crossed with the elav3A-GAL4 line to specifically express Caz double stranded RNA in neuronal tissues. Each independent fly strain carrying elav3A/Caz-IR1-167 showed essentially the same phenotype as the strain carrying Caz-IR363-399/+;elav3A/+. These results suggest that the phenotypes observed in the neuron-specific Caz-knockdown flies were not due to an off-target effect but rather to a reduction in Caz protein levels (Sasayama, 2012).
Mutations in the FUS gene are associated with inherited forms of both ALS and FTLD. The FUS gene was originally identified in a study that found that the FUS protein forms part of a fusion protein with the transcription factor CHOP, which arises due to a chromosomal translocation in liposarcoma. It has been reported that there are both dominantly and recessively inherited families of ALS with FUS mutations. Before the discovery of these FUS mutations in familial ALS, mutations in the TARDBP gene that encodes another RNA-binding protein, TDP-43, had been reported to be associated with familial ALS and FTLD. Both the FUS gene and the TARDBP gene encode an RNA-binding protein equipped with an RRM, and should therefore be involved in RNA processing, splicing, and RNA metabolism. Since FUS and TDP-43 have substantial similarities in their protein structure and putative functions, they could therefore cause ALS or FTLD through common pathogenic processes. However, the mechanisms through which mutations in FUS or TARDBP cause ALS and FTLD are not known, and both toxic gain-of-function and loss-of-function models have been proposed. ALS-associated mutant forms of TDP-43 and FUS are known to form abnormal cytosolic aggregates, and high-level overexpression of either wild-type or mutant TDP-43 is neurotoxic in mice, zebra fish and Drosophila. One recent study reported that a Drosophila model in which targeted expression of mutant human FUS in Drosophila motor neurons led to locomotor dysfunction (Lanson, 2011). These findings would support the toxic gain-of-function model. However, overexpression of mutant proteins may also perturb the activity of endogenous TDP-43, supporting the loss-of-function model. Similarly, the targeted expression model mentioned above reported that deletion of the nuclear export signal rescued toxicity associated with mutant FUS, suggesting that delocalization of FUS from the nucleus to the cytoplasm, namely the loss-of-nuclear-function, would be necessary for neurodegeneration (Sasayama, 2012).
This study has demonstrated that neuron-specific knockdown of Caz, the Drosophila FUS homologue, could induce a defect in fly locomotive abilities as well as degeneration of motoneurons at NMJs in the model flies. There has been one previous report that showed that flies lacking TBPH, the Drosophila TDP-43 homologue, present deficient locomotive behaviors, reduced life span and anatomical defects at the NMJs (Feiguin, 2009). Regarding FUS and its homologues, one recent study reported that Drosophila mutants in which the Caz gene was disrupted exhibited decreased adult viability, diminished locomotor speed and reduced life span compared with controls, and that these phenotypes were fully rescued by wild-type human FUS, but not by ALS-associated mutant FUS (Wang, 2011). These reports, together with the current results, demonstrated that a lack of physiological functions of FUS or TDP-43 in the nucleus is sufficient for induction of locomotive dysfunction and motoneuron degeneration, which recapitulate the phenotypes of ALS, and they therefore imply that the loss of physiological FUS functions are sufficient for the development of pathogenic processes similar to those that occur in FUS- or TDP-43-related ALS/FTLD, in the absence of cytosolic aggregates that may be toxic to motoneurons in ALS/FTLD (Sasayama, 2012).
There have been a few previous studies in which loss-of-function animal models of FUS-related human disorders were generated. FUS knockout mice show perinatal lethality and defects in B lymphocyte development. Additionally, the hippocampal pyramidal neurons of these FUS-null mice exhibited abnormal spine morphology and lower spine density (Fujii, 2005). One report showed that surviving knockout mice exhibited male sterility. However, the neurodegenerative phenotypes of these mice have not been reported to date. With regard to Drosophila models, one recent paper that was mentioned above presented a mutant fly strain (named the Caz1 mutant) in which 58% of the Caz gene was deleted by creating a small genomic deletion (Wang, 2011). This fly model developed a phenotype of disturbed locomotion that is similar to that observed in the Caz-knockdown flies in the present study. The differences between the Caz1 mutant and the fly models of this study were as follows: (1) the Caz1 mutant did not show any morphological abnormalities at the NMJs i.e., shortening of the presynaptic terminals of motoneurons and decrease in the number of synaptic boutons, both of which were observed in the current Caz-knockdown models. (2) The Caz1 mutant showed reduced life spans, which were not observed in the current models, and this life-span defect of Caz1 mutant could be fully rescued by expression of wild-type fly Caz or wild-type human FUS in neurons using elav-Gal4. The difference in life span between the Caz1 mutant and the current Caz-knockdown models might be caused by differences in the expression pattern of the transgenes between the two models; in the current fly models Caz gene expression was knocked down specifically in the nervous system, whereas, in the short-lived Caz1 mutant flies, Caz was disrupted throughout the whole body. In the Caz-knockdown models of this study, the expression of Caz protein was knocked down to 40%-60% in the CNS, but their life spans were not reduced. Together with the fact that the reduced life span of the Caz1 mutant was rescued by the neuronal expression of wild-type Caz, the current results suggest that substantial expression of Caz in neuronal tissues, even though it is not fully expressed, could sufficiently keep their life spans within normal range. The model flies of this study also demonstrated that normal expression of Caz in neurons is essential for the elongation of synaptic branches of motoneurons at NMJs, and therefore that Caz-knockdown would induce impaired maturation of these synaptic branches, resulting in the observed locomotive deficit in the model flies, in the absence of any non-neuronal effect of the Caz protein (Sasayama, 2012).
In conclusion, this study established fly models with neuron-specific knockdown of the Drosophila FUS homologue, and showed that those flies developed locomotive deficits as well as anatomical defects of motoneurons at NMJs. The results indicate that the loss of physiological FUS functions in the nucleus is more likely to be the fundamental pathogenic mechanism that causes FUS-related ALS/FTLD than the toxicity of cytoplasmic aggregates. These data further indicate future research directions, suggesting that it will be necessary to identify target molecules, including nuclear proteins and/or RNA species that associate with FUS, in order to elucidate the molecular mechanisms leading to neuronal dysfunction in FUS-associated ALS/FTLD and to develop the disease-modifying therapies that are eagerly desired in those relentless neurodegenerative diseases. In any event, the Drosophila model that this study established, that recapitulates key features of human ALS, would be suitable for the screening of genes and chemicals that can modify these pathogenic processes that lead to the degeneration of motoneurons in ALS (Sasayama, 2012).
The fatal adult motor neuron disease amyotrophic lateral sclerosis (ALS) shares some clinical and pathological overlap with frontotemporal dementia (FTD), an early-onset neurodegenerative disorder. The RNA/DNA-binding proteins fused in sarcoma (FUS; also known as TLS) and TAR DNA binding protein-43 (TDP-43) have recently been shown to be genetically and pathologically associated with familial forms of ALS and FTD. It is currently unknown whether perturbation of these proteins results in disease through mechanisms that are independent of normal protein function or via the pathophysiological disruption of molecular processes in which they are both critical. This study reports that Drosophila mutants in which the homolog of FUS is disrupted exhibit decreased adult viability, diminished locomotor speed, and reduced life span compared with controls. These phenotypes were fully rescued by wild-type human FUS, but not ALS-associated mutant FUS proteins. A mutant of the Drosophila homolog of TDP-43 had similar, but more severe, deficits. Through cross-rescue analysis, it was demonstrated that FUS acted together with and downstream of TDP-43 in a common genetic pathway in neurons. Furthermore, it was found that these proteins associated with each other in an RNA-dependent complex. These results establish that FUS and TDP-43 function together in vivo and suggest that molecular pathways requiring the combined activities of both of these proteins may be disrupted in ALS and FTD (Wang, 2011).
Amyotrophic lateral sclerosis (ALS) is progressive neurodegenerative disease characterized by the loss of motor function. Several ALS genes have been identified as their mutations can lead to familial ALS, including the recently reported RNA-binding protein fused in sarcoma (Fus). However, it is not clear how mutations of Fus lead to motor neuron degeneration in ALS. This study presents a Drosophila model to examine the toxicity of Fus, its Drosophila orthologue Cabeza (Caz), and the ALS-related Fus mutants. The results show that the expression of wild-type Fus/Caz or FusR521G induced progressive toxicity in multiple tissues of the transgenic flies in a dose- and age-dependent manner. The expression of Fus, Caz, or FusR521G in motor neurons significantly impaired the locomotive ability of fly larvae and adults. The presynaptic structures in neuromuscular junctions were disrupted and motor neurons in the ventral nerve cord (VNC) were disorganized and underwent apoptosis. Surprisingly, the interruption of Fus nuclear localization by either deleting its nuclear localization sequence (NLS) or adding a nuclear export signal (NES) blocked Fus toxicity. Moreover, it was discovered that the loss of caz in Drosophila led to severe growth defects in the eyes and VNCs, caused locomotive disability and NMJ disruption, but did not induce apoptotic cell death. These data demonstrate that the overexpression of Fus/Caz causes in vivo toxicity by disrupting neuromuscular junctions (NMJs) and inducing apoptosis in motor neurons. In addition, the nuclear localization of Fus is essential for Fus to induce toxicity. These findings also suggest that Fus overexpression and gene deletion can cause similar degenerative phenotypes but the underlying mechanisms are likely different (Xia, 2012).
In humans, mutations in the fused in sarcoma (FUS) gene have been identified in sporadic and familial forms of amyotrophic lateral sclerosis (ALS). Cabeza (Caz) is the Drosophila ortholog of human FUS. Previous studies have established Drosophila models of ALS harboring Caz-knockdown. These flies develop locomotive deficits and anatomical defects in motoneurons (MNs) at neuromuscular junctions; these phenotypes indicate that loss of physiological FUS functions in the nucleus can cause MN degeneration similar to that seen in FUS-related ALS. This study aimed to explore molecules that affect these ALS-like phenotypes of Drosophila models with eye-specific and neuron-specific Caz-knockdown. Several previously reported ALS-related genes were examined, and genetic links were found between Caz and ter94, the Drosophila ortholog of human Valosin-containing protein (VCP). Genetic crossing the strongest loss-of-function allele of ter94 with Caz-knockdown strongly enhanced the rough-eye phenotype and the MN-degeneration phenotype caused by Caz-knockdown. Conversely, the overexpression of wild-type ter94 in the background of Caz-knockdown remarkably suppressed those phenotypes. These data demonstrated that expression levels of Drosophila VCP ortholog dramatically modified the phenotypes caused by Caz-knockdown in either direction, exacerbation or remission. These results indicate that therapeutic agents that up-regulate the function of human VCP could modify the pathogenic processes that lead to the degeneration of MNs in ALS (Azuma, 2014).
VCP is a member of the AAA (ATPase associated with a variety of cellular activities) family of proteins, which are implicated in a large variety of biological functions including the regulation of ubiquitin-dependent protein degradation, control of membrane fusion and of dynamics of subcellular components, vesicle-mediated transport and nucleocytoplasmic shuttling. Association of VCP mutations with human disease was first identified in patients with IBMPFD (inclusion body myopathy with early-onset Paget disease and frontotemporal dementia) and more recently in those with ALS . There is a single ortholog of human VCP in Drosophila, named ter94, which is predicted to share ∼83% amino acid sequence identity with human VCP (Azuma, 2014).
This study found that genetic crossing the strongest loss-of-function allele of ter94 with Caz-knockdown severely enhanced the Caz-knockdown phenotypes in flies; it severely exacerbated locomotive disabilities and the degeneration of MNs induced by neuron-specific Caz-knockdown. Conversely, the overexpression of ter94 rescued those phenotypes (Azuma, 2014).
These results demonstrate, for the first time, a genetic link between Caz and ter94, the Drosophila orthologs of FUS and VCP, respectively. Although it would be necessary to confirm whether that is Drosophila-specific or not, the results suggest genetic interaction between FUS and VCP in human. Genetic interaction between TDP-43 and VCP in Drosophila was demonstrated previously; IBMPFD-causing mutations in ter94 lead to redistribution of TDP-43, from the nucleus to the cytoplasm, and redistribution of TDP-43 is sufficient to induce morphologically aberrant rough eyes (Ritson, 2010). This previous report suggests that VCP can balance the amount of TDP-43, which is a constituent of larger heteronuclear ribonucleoprotein (hnRNP) complexes, between nucleus and cytoplasm by acting as a nucleocytoplasmic shuttling molecule. In this schema, VCP functions to remove TDP-43 from RNP complexes, import TDP-43 into nuclei and degrade TDP-43 via autophagy. VCP might have similar functions with respect to FUS because FUS and TDP-43 have significant structural and functional similarities and are implicated in similar molecular processes. For example, TDP-43 and FUS act in the context of larger hnRNP complexes. FUS also continuously moves between the nucleus and the cytoplasm; therefore, FUS not only regulates gene expression in the nucleus, but also has important functions in the cytoplasm. This study showed that the decreased level of Caz in the nucleus and the resultant motor disturbance induced by neuron-specific Caz-knockdown could be rescued by overexpressed wild-type ter94 despite lacking any change of Caz protein in the CNS. If VCP has a shuttling function, wild-type ter94 overexpression could translocate Caz from cytoplasm to nucleus because nuclear importing function of ter94 would be dominantly induced in the situation with the deficiency of Caz in the nucleus. Conversely, the loss-of-function allele of ter94 (ter94k15502) exacerbated the depletion of Caz from the nucleus probably because ter94-mediated nuclear import of Caz was compromised (Azuma, 2014).
It has been demonstrated that a polyQ tract can interact with VCP in Drosophila; specifically, either the strongest (ter94k15502) or strong (ter9403775) loss-of-function allele of ter94 suppressed the eye degeneration induced by an expanded polyQ tract, whereas the overexpression of wild-type ter94 in the background of Caz-knockdown enhanced this phenotype. Additionally, a chromosomal deletion of 46C3-46E02, the genomic region that contains ter94, acted as a dominant suppressor of the polyQ-induced phenotype. The present study and these previous reports together indicate that gain and loss of ter94 function rescued and exacerbated Caz-knockdown phenotypes, respectively, and that they had the converse effects on polyQ-induced phenotypes. These converse effects could be explained by the difference in disease pathogenesis; in polyQ-induced disease models, polyQ-containing pathogenic aggregates exist in nuclei of affected neurons; in contrast, Caz expression in nuclei is deficient in Caz-knockdown disease models. Overexpression of wild-type ter94, which functions in nuclear import of polyQ or Caz, would exacerbate nuclear polyQ aggregation, but could alleviate the nuclear deficiency of Caz protein (Azuma, 2014).
Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that causes progressive muscular weakness. Fused in Sarcoma (FUS) that has been identified in familial ALS is an RNA binding protein that is normally localized in the nucleus. However, its function in vivo is not fully understood. Drosophila Cabeza (Caz) is a FUS homologue and specific knockdown of Caz in the eye imaginal disc and pupal retina using a GMR-GAL4 driver was found to induce an abnormal morphology of the adult compound eyes, a rough eye phenotype. This was partially suppressed by expression of the apoptosis inhibitor P35. Knockdown of Caz exerted no apparent effect on differentiation of photoreceptor cells. However, immunostaining with an antibody to Cut that marks cone cells revealed fusion of these and ommatidia of pupal retinae. These results indicate that Caz knockdown induces apoptosis and also inhibits differentiation of cone cells, resulting in abnormal eye morphology in adults. Mutation in EGFR pathway-related genes, such as rhomboid-1, rhomboid-3 and mirror, suppresses the rough eye phenotype induced by Caz knockdown. Moreover, the rhomboid-1 mutation rescues the fusion of cone cells and ommatidia observed in Caz knockdown flies. The results suggest that Caz negatively regulates the EGFR signaling pathway required for determination of cone cell fate in Drosophila (Shimamura, 2014).
This study found that Caz knockdown in eye imaginal discs induces a rough eye phenotype associated with apoptosis, abnormal differentiation of cone cells and pigment cells, and defects in ommatidia rotation in pupal retinae. However, apoptosis and differentiation of photoreceptor cells were not affected in larval eye imaginal discs expressing Caz dsRNA. Why did Caz knockdown in eye imaginal discs affect pupal retinae but not third instar larval eye discs? In situ hybridization and immunohistochemistrical analyses demonstrated that Caz mRNA and protein are enriched in the brain and CNS during embryogenesis, and Caz protein was detected in the nuclei of several larval tissues and in imaginal discs. However, the expression level of Caz is higher in adult eyes than in larval eye discs (Flybase). Thus, it is possible that Caz plays a more important role in eye development in the pupal stage (Shimamura, 2014).
The observation that the rough eye phenotype of Caz knockdown flies was significantly suppressed by co-expression of P35 and that apoptotic cells detected by immunostaining with anti-cleaved Caspase-3 antibody were significantly increased in pupal retinae of flies expressing Caz dsRNA suggests that induction of apoptosis at least partially contributes to the rough eye phenotype. It is reported that the number of dying cells increases dramatically if interactions between cells are disrupted, for instance upon cell ablation. Therefore, one possible explanation is that Caz knockdown disrupts interactions between cells in pupal retinae, as evidenced with anti-Cut immunostaining, that results in induction of apoptosis. In addition, it is well known that apoptosis is induced by JNK or p38 signaling. It is also reported that persistent activation of the JNK or p38 signaling pathways mediates neuronal apoptosis in ALS, and that TDP-43 is related to JNK signaling. Thus, another possible explanation is that Caz knockdown induces JNK or p38 signaling, resulting in increase of apoptosis in pupal retinae (Shimamura, 2014).
This study found a genetic interaction between Caz and Rhomboid, a rate-limiting component of the EGFR signaling pathway. Appropriate levels of EGFR signaling are required for cone cell-fate and ommatidial rotation. Knockdown of Caz in eye imaginal discs and pupal retinae induced abnormal differentiation of cone cells and defects in ommatidia rotation that eventually resulted in the rough eye phenotype in adults. The rhomboid-1 mutant rescued the fusion of cone cells and mutations of rhomboid-3 and mirror significantly suppressed the rough eye phenotype of Caz knockdown flies. In contrast, mutations of EGFR did not suppress the rough eye phenotype induced by knockdown of Caz. These apparently contradictory results might be explained as follows. Once activated, the signaling cascade could be amplified progressively, so that only a half reduction of some components of pathway such as EGFR may not be sufficient to suppress the effects of over-activation of the initiator such as Rhomboid. In any event, the present study suggests that Caz negatively regulates EGFR signaling. Since the expression level of Caz is much higher in adult eyes than larval eye discs, negative regulation of EGFR signaling by Caz may play a role in controlling EGFR signaling less reactive to oxidative stress during adulthood. It should be noted that a hallmark of ALS is chronic neuronal exposure to oxidative stress and inflammation (Shimamura, 2014).
In summary, this study has shown that knockdown of Caz in the Drosophila retina induces a rough eye phenotype associated with increased apoptosis, abnormal differentiation of cone cells and pigment cells, and defects in ommatidia rotation. This study provides the first definitive evidence that Caz plays an important role in regulation of the EGFR signaling pathway. It should be noted that the neurodegeneration occurring in ALS can be accounted for deviation from strict control of MAPK signaling. Thus, the Caz knockdown flies used in the present study should provide a useful tool for elucidating functions of FUS and pathological mechanisms of associated ALS (Shimamura, 2014).
Mutations in the RNA binding protein Fused in sarcoma (FUS) are estimated to account for 5-10% of all inherited cases of amyotrophic lateral sclerosis (ALS), but the function of FUS in motor neurons is poorly understood. This study investigated the early functional consequences of overexpressing wild-type or ALS-associated mutant FUS proteins in Drosophila motor neurons, and compare them to phenotypes arising from loss of the Drosophila homolog of FUS, Cabeza (Caz). Lethality and locomotor phenotypes were found to correlate with levels of FUS transgene expression, indicating that toxicity in developing motor neurons is largely independent of ALS-linked mutations. At the neuromuscular junction (NMJ), overexpression of either wild-type or mutant FUS results in decreased number of presynaptic active zones and altered postsynaptic glutamate receptor subunit composition, coinciding with a reduction in synaptic transmission as a result of both reduced quantal size and quantal content. Interestingly, expression of human FUS downregulates endogenous Caz levels, demonstrating that FUS autoregulation occurs in motor neurons in vivo. However, loss of Caz from motor neurons increases synaptic transmission as a result of increased quantal size, suggesting that the loss of Caz in animals expressing FUS does not contribute to motor deficits. These data demonstrate that FUS/Caz regulates NMJ development and plays an evolutionarily conserved role in modulating the strength of synaptic transmission in motor neurons (Machamer, 2014).
Several models of FUS-mediated neurodegeneration have been developed in Drosophila and other organisms), but the pathogenic mechanisms underlying their phenotypes are poorly understood. Toxicity in these models can arise from overexpression of wild-type FUS alone, independent of any disease-associated mutations. Dissecting the effects of disease-associated mutations when wild-type expression alone confers toxicity is difficult and requires carefully controlled levels of gene expression. A previous study concluded that ALS-linked mutations in FUS resulted in a toxic gain-of-function in Drosophila, and this conclusion was based on the finding that the wild-type and mutant UAS-HA-tagged FUS (UAS-HA-FUS) transgenic lines expressed equivalent levels of protein in the eye. However, this analysis of these same UAS-HA-FUS lines demonstrates that when expressed in motor neurons, message and protein expression are 3- to 4-fold higher in the R521C mutant line than the wild-type line. Thus, these results suggest that the increased severity of phenotypes seen in the mutant line relative to wild-type is due to increased expression level rather than mutation-specific toxicity. Therefore, no evidence of a gain-of-function effect of ALS-associated mutations in FUS was found in this model. These results are further supported by another study using independently generated transgenic lines that demonstrated that FUS-mediated overexpression toxicity in the adult eye was independent of ALS pathogenic mutations (Machamer, 2014).
There are two non-mutually exclusive explanations for these dramatic differences in relative transgene expression levels observed in different tissues. First, given that HA-FUSR521C overexpression in eyes causes degeneration, the simplest explanation is that HA-FUSP525C lines express at higher levels than wild-type in the eyes during development, and this leads to cell loss or dysfunction that causes a reduction in protein expression in the adult, whereas expression in glutamatergic neurons with OK371-GAL4 does not have these effects. Indeed, morphological analysis of GMR>FUSR524C fly eyes demonstrates severe cell loss. An alternative possibility is that tissue-specific enhancers may differentially regulate gene expression from P elements located at different genomic loci, as these position effects are well known in Drosophila. These observations suggest two important guidelines for analyzing overexpression disease models in Drosophila. First, given the widely available technologies for site-specific transgenesis, comparisons of gain-of-function phenotypes between wild-type and mutant proteins should utilize transgenic lines inserted at identical genomic sites. Indeed, when lines generated in this manner (FL-FUS) were analyzed, identical expression levels were seen between the wild-type and mutant proteins in motor neurons. Second, when comparing protein expression levels between wild-type and mutant transgenic lines, expression levels should be compared in the absence of cell loss and in the tissue most relevant to the disease (Machamer, 2014).
Recently, FUS has been shown to autoregulate its expression levels in HeLa cells by binding to exon 7 and flanking introns of its own pre-mRNA (Zhou, 2013). Interestingly, ALS-associated mutations in the C-terminus of FUS have been suggested to disrupt autoregulation, and mutations in the 3'UTR of human FUS that cause FUS overexpression have been shown to cause ALS. Together, these data suggest that disruption of FUS autoregulation leading to overexpression of wild-type FUS is sufficient to cause disease. This study shows that wild-type or mutant FUS overexpression in Drosophila motor neurons leads to severe (~90%) downregulation of fly FUS. This suggests that an autoregulatory mechanism is conserved in Drosophila and occurs in motor neurons in vivo. This autoregulation likely explains why in many cases, stronger phenotypes are not seen in higher-expressing transgenic lines. Future studies will investigate the mechanism of this autoregulation and the effect of ALS-causing disease mutations in this model (Machamer, 2014).
Whether disease-associated mutations in FUS and other ALS genes cause disease through a gain- or loss-of-function is a matter of debate. Simple genetic model systems such as Drosophila are ideal for interpreting the effect of mutations on protein function; however, one must be cautious in interpreting results gained from overexpression studies, particularly when overexpressing human proteins in the fly. However, because low-level expression of human FL-FUS in neurons rescues phenotypes caused by loss of Drosophila FUS (i.e., Caz) (Wang, 2011), this strongly argues for evolutionary conservation of FUS as well as a requirement for FUS in neurons. Furthermore, because FUSP525L fails to rescue Caz phenotypes, this strongly argues that the P525L mutation causes a loss-of-function. Consistent with this interpretation, analyses of disease-associated mutations in FUS are most consistent with a partial loss-of-function, given that (1) low-level FUSWT but not FUSP525L expression causes increased larval and adult locomotor activity, and (2) expressing higher level FUSP525C in some instances causes less severe phenotypes than lower level FUSWT expression (e.g. Caz downregulation), altered EJP rise time, and GluRIIA upregulation. Nonetheless, the possibility cannot be excluded that FUS mutations do exhibit some gain-of-toxic effects, particularly given that a greater reduction in GluRIIB levels is seen with mutant FUS expression than with wild-type expression. Since mutations in the 3'UTR autoregulatory domain are sufficient to cause ALS in patients by upregulating wild-type FUS protein, ALS may be caused by increased levels of wild-type protein. In this context, low-level overexpression of FUS or Caz in aging flies may be a reasonable way to model the disease (Machamer, 2014).
FUS has been implicated in a wide range of processes in many cell types both within the nucleus and cytoplasm. Since it was not possible to detect significant wild-type FUS or Caz protein within motor axons or at the NMJ, it is postulated that FUS/Caz normally functions in the nucleus to regulate the expression of genes that modulate synapse function. Importantly, FUS/Caz appears to be required within neurons to regulate synaptic transmission, as caz1 loss-of-function phenotypes are mimicked by presynaptic caz knockdown, and overexpression of FUS in motor neurons leads to altered synaptic transmission (Machamer, 2014).
This study shows that FUS expression inhibits evoked release due to both a reduction of quantal size (mEJP amplitude) and quantal content (number of quanta released per stimulus). A reduction in mEJP amplitude can be due to a decrease in synaptic vesicle size, glutamate concentration or postsynaptic currents due to alterations in glutamate receptor levels, localization or composition. It is postulated that the reduction in quantal size is due to a disruption of the spatial coupling of synaptic vesicle release sites with glutamate receptors given the disruption in the number and morphology of active zones (labeled with Brp) and the postsynaptic density (labeled with Dlg). Importantly, this study demonstrates that reduced GluR levels are not responsible for the decrease in mEJP amplitude observed in FUS overexpressing animals, but rather that GluR clustering at active zones may be altered. Furthermore, an increase is seen in relative expression of A-type GluRs which would be expected to have the opposite effect on mEJP amplitude. This increase in ratio of A- to B-type GluRs is seen when glutamate release is blocked at larval NMJs and is a homeostatic response to reduction in glutamate-mediated synaptic transmission (Machamer, 2014).
Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease that leads invariably to fatal paralysis associated with motor neuron degeneration and muscular atrophy. One gene associated with ALS encodes the DNA/RNA-binding protein Fused in Sarcoma (FUS). There now exist two Drosophila models of ALS. In one, human FUS with ALS-causing mutations is expressed in fly motor neurons; in the other, the gene cabeza (caz), the fly homolog of FUS, is ablated. These FUS-ALS flies exhibit larval locomotor defects indicative of neuromuscular dysfunction and early death. The locus and site of initiation of this neuromuscular dysfunction remain unclear. This study shows that in FUS-ALS flies, motor neuron cell bodies fire action potentials that propagate along the axon and voltage-dependent inward and outward currents in the cell bodies are indistinguishable in wild-type and FUS-ALS motor neurons. In marked contrast, the amplitude of synaptic currents evoked in the postsynaptic muscle cell is decreased by >80% in FUS-ALS larvae. Furthermore, the frequency but not unitary amplitude of spontaneous miniature synaptic currents is decreased dramatically in FUS-ALS flies, consistent with a change in quantal content but not quantal size. Although standard confocal microscopic analysis of the larval neuromuscular junction reveals no gross abnormalities, superresolution stimulated emission depletion (STED) microscopy demonstrates that the presynaptic active zone protein Bruchpilot is aberrantly organized in FUS-ALS larvae. The results are consistent with the idea that defects in presynaptic terminal structure and function precede, and may contribute to, the later motor neuron degeneration that is characteristic of ALS (Shahidullah, 2013).
Communication between motor neurons and their target muscles is compromised in amyotrophic lateral sclerosis (ALS). Although overt symptoms in humans and mouse models are associated with muscle atrophy after muscle denervation, the primary defects that precipitate this loss of neuromuscular connectivity remain poorly understood. Most of what is known about the degradation of the neuromuscular synapse in ALS comes from pathological studies in mouse models of superoxide dismutase 1 (SOD1)-mediated ALS. Analysis of disease progression in these mice suggests that ALS starts in the terminal axon, leading to muscle dysfunction and denervation, and proceeds in a retrograde pattern, with degeneration of the proximal motor axon and eventual loss of neuronal cell bodies. This implies that a local defect in the distal portion of the axon and/or the presynaptic terminal is a primary pathogenic event in disease progression. However, little is known about intrinsic changes in mammalian motor neuron excitability and synaptic transmission in ALS, in part because of the challenges of performing electrophysiological recordings in vivo (Shahidullah, 2013).
Mutations in the DNA/RNA-binding protein Fused in Sarcoma (FUS), as well as in other DNA/RNA-binding proteins such as TDP43, have been implicated in both familial and sporadic cases of ALS (Sreedharan, 2008: Kwiatkowski, 2009; Vance, 2009). Recently, models of both FUS-related and TDP43-related ALS have been established both in Drosophila (Lanson, 2011; Wang, 2011; Miguel, 2012; Sasayama, 2012; Xia, 2012) and in zebrafish (Kabashi, 2010; Armstrong, 2013a; Armstrong, 2013b). Expression of human disease-causing mutants of either FUS or TDP43 in flies or fish leads to impaired locomotor activity and early death (Kabashi, 2010; Lanson, 2011). Flies that lack the gene cabeza (caz), the fly homolog of FUS, exhibit similar phenotypes that can be rescued by the expression of wild-type but not mutant human FUS (Wang, 2011), consistent with the idea that FUS-mediated ALS involves both loss of nuclear function and gain of toxic function from accumulation of mislocalized cytoplasmic mutant FUS. In the fly, because cell-type-specific drivers are used to restrict the expression of mutant human FUS to motor neurons, it is evident that the locomotor and life span phenotypes must arise from defects in the presynaptic cell. Morphological and electrophysiological analysis of the fish neuromuscular junction also suggests that presynaptic changes accompany the disease phenotype (Shahidullah, 2013).
This study has examined motor neuron cell body excitability, axonal conduction, synaptic transmission, and synapse structure in mutant FUS-expressing and cabeza-null flies. Neuronal excitability and action potential propagation are essentially normal at the same time that neurotransmitter release is severely impaired and presynaptic active zone structure is aberrant. The results suggest that defects in presynaptic structure and function are early and are perhaps precipitating events in the pathogenesis of FUS-related ALS (Shahidullah, 2013).
The demonstration that mutations in the DNA/RNA-binding proteins TDP43 and FUS are associated with both familial and sporadic ALS has profoundly influenced thinking about the pathogenesis of the disease. It is becoming evident that the impact of these findings will not be restricted to ALS, because mutations in these same proteins are implicated in at least one other neurodegenerative disorder, frontotemporal lobar degeneration. Despite the enormous interest generated by these findings, the mechanisms by which TDP43 and FUS lead to disease onset and progression remain poorly understood. Both are multifunctional proteins that participate in transcriptional regulation and in several aspects of mRNA processing, splicing, transport, and perhaps local translation at the synapse and they may bind to and influence noncoding RNAs as well (Polymenidou, 2012; Shahidullah, 2013 and references therein).
This study shows that the motor neuron cell bodies of R521C-FUS-expressing larvae generate normal voltage-dependent inward and outward currents and fire action potentials in response to depolarizing stimuli. Furthermore, these action potentials can propagate normally along the axon. Indeed, if anything, the FUS-ALS motor neurons are somewhat more excitable than neurons that express wild-type FUS, again in agreement with the results in zebrafish and prior findings in SOD1 mice. The biophysical mechanism of this widely observed increase in motor neuron excitability in ALS models remains to be determined (Shahidullah, 2013).
Despite the fact that motor neuron cell body and axon excitability are normal or enhanced, this study found that synaptic function at the larval neuromuscular junction is profoundly impaired in flies that overexpress R521C-FUS, as well as in caz-null flies. Evoked synaptic transmission is decreased by >80% and spontaneous mEJC frequency is also reduced substantially. These findings reinforce those reported for the zebrafish larval neuromuscular junction after expression of mutant human TDP43 or FUS. In the fly, although it is uncertain whether the effect of caz knock-out is pre- or postsynaptic, the changes observed in mEJC frequency strongly suggest a major presynaptic contribution; a similar inference has been drawn in the zebrafish. Furthermore, in the fly, there is no uncertainty about the primary presynaptic locus in the case of R521C-FUS expression because the OK-371GAL4 driver restricts expression to the presynaptic motor neuron and, again, mEJC frequency is decreased substantially. Quantal analysis reveals that the unitary mEJC amplitude is the same in R521C-FUS-expressing and caz1 larvae as in larvae expressing wild-type FUS. This lack of change in quantal size suggests that the size and neurotransmitter content of synaptic vesicles is not affected by FUS genotype. Rather, the decrease in evoked synaptic transmission in the FUS-ALS larvae is most readily explained by a decrease in quantal content, the number of vesicles released in response to a given stimulus. Such a decrease in evoked release probability is consistent with the decrease in spontaneous mEJC frequency observed in the FUS-ALS larvae (Shahidullah, 2013).
It is surprising and intriguing that the decay of the evoked EJC is considerably faster in the R521C-FUS and caz1 larvae. Such a change in the kinetics of the EJC might be consistent with a change in the deactivation properties of the postsynaptic glutamate receptors, perhaps secondary to the initial presynaptic defect. Conversely, another possibility is that presynaptic glutamate uptake systems are among the many targets of FUS and are altered in R521C-FUS-expressing and caz1 motor neurons and that this accounts for the changes in EJC kinetics. A detailed analysis of glutamate receptor properties and glutamate diffusion and reuptake will be required to distinguish between these interesting possibilities and to explain how the kinetics of the evoked EJC are altered by FUS genotype whereas those of the unitary mEJC are not (Shahidullah, 2013).
Examination of presynaptic bouton structure by confocal microscopy did not reveal any gross morphological abnormalities in the present study or in Lanson (2011) (but see Xia, 2012). In contrast, using STED microscopy, this study showed that the presynaptic active zones, measured by staining the active zone protein Bruchpilot, are misshapen and disorganized in R521C-FUS larvae. Although Bruchpilot itself is essential for the formation of active zones and neurotransmitter release, this study used it as a marker of active zone organization, as has been done previously. Misshapen bruchpilot-labeled active zones like those observed in R521C-FUS-expressing larvae are associated with impaired calcium channel clustering and a decrease in calcium influx (Liu, 2011) and, in the zebrafish, treatment with calcium channel agonists can rescue both locomotor and synaptic transmission defects induced by expression of mutant TDP43 (Armstrong, 2013a). It will be interesting to use STED microscopy to examine calcium channel organization at active zones in FUS-aberrant Drosophila larvae (Shahidullah, 2013).
The results of this study demonstrate clearly that structural abnormalities at the synapse and defects in neurotransmitter release precede motor neuron cell body and axonal degeneration in two fly models of FUS-related ALS. Many questions remain to be explored, including the identity of the critical FUS target(s), the mechanism by which overexpression of mutant FUS and knock-out of endogenous caz both produce the same synaptic phenotype, and the way that synaptic dysfunction leads ultimately to motor neuron degeneration. These findings and those in the zebrafish point to the importance of early therapeutic intervention at the synapse in developing novel treatments for ALS (Shahidullah, 2013).
Autosomal dominant mutations of the RNA/DNA binding protein FUS are linked to familial amyotrophic lateral sclerosis (FALS); however, it is not clear how FUS mutations cause neurodegeneration. Using transgenic mice expressing a common FALS-associated FUS mutation (FUS-R521C mice), this study found that mutant FUS proteins formed a stable complex with WT FUS proteins and interfered with the normal interactions between FUS and histone deacetylase 1 (HDAC1). Consequently, FUS-R521C mice exhibited evidence of DNA damage as well as profound dendritic and synaptic phenotypes in brain and spinal cord. To provide insights into these defects, this study screened neural genes for nucleotide oxidation and identified brain-derived neurotrophic factor (Bdnf) as a target of FUS-R521C-associated DNA damage and RNA splicing defects in mice. Compared with WT FUS, mutant FUS-R521C proteins formed a more stable complex with Bdnf RNA in electrophoretic mobility shift assays. Stabilization of the FUS/Bdnf RNA complex contributed to Bdnf splicing defects and impaired BDNF signaling through receptor TrkB. Exogenous BDNF only partially restored dendrite phenotype in FUS-R521C neurons, suggesting that BDNF-independent mechanisms may contribute to the defects in these neurons. Indeed, RNA-seq analyses of FUS-R521C spinal cords revealed additional transcription and splicing defects in genes that regulate dendritic growth and synaptic functions. Together, these results provide insight into how gain-of-function FUS mutations affect critical neuronal functions (Qiu, 2014; full text of article).
The gene encoding a DNA/RNA binding protein FUS/TLS is frequently mutated in amyotrophic lateral sclerosis (ALS). Mutations commonly affect its carboxy-terminal nuclear localization signal, resulting in varying deficiencies of FUS nuclear localization and abnormal cytoplasmic accumulation. Increasing evidence suggests deficiencies in FUS nuclear function may contribute to neuron degeneration. This study reports a novel FUS autoregulatory mechanism and its deficiency in ALS-associated mutants. Using FUS CLIP-seq, significant FUS binding was identified to a highly conserved region of exon 7 and the flanking introns of its own pre-mRNAs. FUS was demonstrated to be a repressor of exon 7 splicing; the exon 7-skipped splice variant is subject to nonsense-mediated decay (NMD). Overexpression of FUS led to the repression of exon 7 splicing and a reduction of endogenous FUS protein. Conversely, the repression of exon 7 was reduced by knockdown of FUS protein, and moreover, it was rescued by expression of EGFP-FUS. This dynamic regulation of alternative splicing describes a novel mechanism of FUS autoregulation. Given that ALS-associated FUS mutants are deficient in nuclear localization, whether cells expressing these mutants would be deficient in repressing exon 7 splicing was examined. FUS harbouring R521G, R522G or DeltaExon15 mutation (minor, moderate or severe cytoplasmic localization, respectively) were shown to directly correlate with respectively increasing deficiencies in both exon 7 repression and autoregulation of its own protein levels. These data suggest that compromised FUS autoregulation can directly exacerbate the pathogenic accumulation of cytoplasmic FUS protein in ALS. Exon 7 skipping can be induced by antisense oligonucleotides targeting its flanking splice sites, indicating the potential to alleviate abnormal cytoplasmic FUS accumulation in ALS. Taken together, FUS autoregulation by alternative splicing provides insight into a molecular mechanism by which FUS-regulated pre-mRNA processing can impact a significant number of targets important to neurodegeneration (Zhou, 2013).
Inclusions containing Fused in Sarcoma (FUS) are found in familial and sporadic cases of the incurable progressive motor neuron disease amyotrophic lateral sclerosis and in a common form of dementia, frontotemporal dementia. Most disease-associated mutations are located in the C-terminal proline-tyrosine nuclear localization sequence (PY-NLS) of FUS and impair its nuclear import. It has been shown in cell culture that the nuclear import of FUS is mediated by transportin, which binds the PY-NLS and the last arginine/glycine/glycine-rich (RGG) domain of FUS. Methylation of this last RGG domain by protein arginine methyltransferases (PRMTs) weakens transportin binding and therefore impairs nuclear translocation of FUS. To investigate the requirements for the nuclear import of FUS in an in vivo model, different transgenic Drosophila lines were generated expressing human FUS wild type (hFUS wt) and two disease-related variants P525L and R495X, in which the NLS is mutated or completely absent, respectively. To rule out effects caused by heterologous hFUS expression, the corresponding variants for the Drosophila FUS orthologue Cabeza (Caz wt, P398L, Q349X). Expression of these variants in eyes and motor neurons confirmed the PY-NLS-dependent nuclear localization of FUS/Caz and caused neurodegenerative effects. Surprisingly, FUS/Caz toxicity was correlated to the degree of its nuclear localization in this overexpression model. High levels of nuclear FUS/Caz became insoluble and reduced the endogenous Caz levels, confirming FUS autoregulation in Drosophila. RNAi-mediated knockdown of the two transportin orthologues interfered with the nuclear import of FUS/Caz and also enhanced the eye phenotype. Finally, the Drosophila PRMT proteins (DART1-9) were screened, and it was found that knockdown of Dart1 led to a reduction in methylation of hFUS P525L and aggravated its phenotype. These findings show that the molecular mechanisms controlling the nuclear import of FUS/Caz and FUS autoregulation are conserved between humans and Drosophila. In addition to the well-known neurodegenerative effects of FUS loss-of function, these data suggest toxic potential of overexpressed FUS in the nucleus and of insoluble FUS (Jackel, 2014).
Defects in DNA repair have been extensively linked to neurodegenerative diseases, but the exact mechanisms remain poorly understood. This study found that FUS, an RNA/DNA-binding protein that has been linked to amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration, is important for the DNA damage response (DDR). The function of FUS in DDR involved a direct interaction with histone deacetylase 1 (HDAC1), and the recruitment of FUS to double-stranded break sites was important for proper DDR signaling. Notably, FUS proteins carrying familial ALS mutations were defective in DDR and DNA repair and showed a diminished interaction with HDAC1. Moreover, increased DNA damage was observed in human ALS patients harboring FUS mutations. These findings suggest that an impaired DDR and DNA repair may contribute to the pathogenesis of neurodegenerative diseases linked to FUS mutations (Wang, 2013).
Recently, the fused in sarcoma/translated in liposarcoma (FUS) protein has been identified as a major constituent of nuclear and/or cytoplasmic ubiquitin-positive inclusions in patients with frontotemporal lobar degeneration or amyotrophic lateral sclerosis. The molecular mechanisms underlying FUS toxicity are currently not understood. To address aspects of FUS pathogenesis in vivo, new Drosophila transgenic models expressing a full-length wild-type isoform of human FUS protein were generated. It was found that when expressed in retinal cells, FUS proteins are mainly recovered as soluble forms, and their overexpression results in a mild eye phenotype, with malformed interommatidial bristles and the appearance of ectopic extensions. On the other hand, when FUS proteins are specifically targeted to adult differentiated neurons, they are mainly recovered as insoluble forms, and their overexpression drastically reduces fly life span. Importantly, FUS neurotoxicity occurs regardless of inclusion formation. Lastly, this study showed that molecular chaperones reduce FUS toxicity by modulating protein solubility. Altogether, these data indicate that accumulation of insoluble non-aggregated FUS forms might represent the primary toxic species in human FUS proteinopathies (Miguel, 2012).
Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disorder characterized by the loss of motor neurons. Fused in sarcoma/translated in liposarcoma (FUS/TLS) and TAR DNA-binding protein (TDP)-43 are DNA/RNA-binding proteins found to be mutated in sporadic and familial forms of ALS. Ectopic expression of human ALS-causing FUS/TLS mutations in Drosophila caused an accumulation of ubiquitinated proteins, neurodegeneration, larval-crawling defect and early lethality. Mutant FUS/TLS localized to both the cytoplasm and nucleus, whereas wild-type FUS/TLS localized only to the nucleus, suggesting that the cytoplasmic localization of FUS/TLS is required for toxicity. Furthermore, this study found that deletion of the nuclear export signal strongly suppressed toxicity, suggesting that cytoplasmic localization is necessary for neurodegeneration. Interestingly, it was observed that FUS/TLS genetically interacts with TDP-43 in a mutation-dependent fashion to cause neurodegeneration in vivo. In summary, this study demonstrated that ALS-associated mutations in FUS/TLS cause adult-onset neurodegeneration via a gain-of-toxicity mechanism that involves redistribution of the protein from the nucleus to the cytoplasm and is likely to involve an interaction with TDP-43 (Lanson, 2011).
Neuronal dendrites, together with dendritic spines, exhibit enormously diverse structure. Selective targeting and local translation of mRNAs in dendritic spines have been implicated in synapse remodeling or synaptic plasticity. The mechanism of mRNA transport to the postsynaptic site is a fundamental question in local dendritic translation. TLS (translocated in liposarcoma), previously identified as a component of hnRNP complexes, unexpectedly showed somatodendritic localization in mature hippocampal pyramidal neurons. In the present study, TLS was translocated to dendrites and was recruited to dendrites not only via microtubules but also via actin filaments. In mature hippocampal pyramidal neurons, TLS accumulated in the spines at excitatory postsynapses upon mGluR5 activation, which was accompanied by an increased RNA content in dendrites. Consistent with the in vitro studies, TLS-null hippocampal pyramidal neurons exhibited abnormal spine morphology and lower spine density. Our results indicate that TLS participates in mRNA sorting to the dendritic spines induced by mGluR5 activation and regulates spine morphology to stabilize the synaptic structure (Fujii, 2005).
Many oncogenes associated with human sarcomas are composed of a fusion between transcription factors and the N-terminal portions of two similar RNA-binding proteins, TLS and EWS. Though the oncogenic fusion proteins lack the RNA-binding domain and do not bind RNA, the contribution from the N-terminal portion of the RNA-binding protein is essential for their transforming activity. TLS and EWS associate in vivo with RNA polymerase II (Pol II) transcripts. To learn more about the target gene specificity of this interaction, the localization of a Drosophila melanogaster protein that has extensive sequence identity to the C-terminal RNA-binding portions of TLS and EWS was studied in preparations of Drosophila polytene nuclei. cDNA clones encoding the full-length Drosophila TLS-EWS homolog, SARFH (stands for sarcoma-associated RNA-binding fly homolog), were isolated. Functional similarity to TLS and EWS was revealed by the association of SARFH (Cabeza) with Pol II transcripts in mammalian cells and by the ability of SARFH to elicit homologous down-regulation of the levels of the mammalian proteins. The SARFH gene is expressed in the developing Drosophila embryo from the earliest stages of cellularization and is subsequently found in many cell types. In preparations of polytene chromosomes from salivary gland nuclei, SARFH antibodies recognize their target associated with the majority of active transcription units, revealed by colocalization with the phosphorylated form of RNA Pol II. It is concluded that SARFH and, by homology, EWS and TLS participate in a function common to the expression of most genes transcribed by RNA Pol II (Immanuel, 1995).
A partial Drosophila cDNA, clone P19, has been described that bears homology to members of the RNA recognition motif (RRM) family of proteins. RNA binding as well as involvement in RNA processing has been demonstrated for some RRM proteins. This study reports the further characterization of P19, which was renamed cabeza (caz). caz is located on the X chromosome at position 14B. Using Northern analysis, at least four transcripts from the caz gene were observed at varying levels during development. caz mRNA and protein are enriched in the brain and central nervous system during embryogenesis. In addition, the protein is enriched in the adult head. UV crosslinking was used to demonstrate in vitro RNA binding activity for full-length recombinant caz protein and for the caz RRM domain. Sequence analysis revealed caz is related to two human genes, EWS and TLS, which are involved in chromosomal translocations. The fusion of EWS and TLS to other cellular genes results in sarcoma formation. In addition to their overall structural organization and sequence similarity, these three genes share an RRM which is divergent from typical RRMs. Therefore, it appears that these genes constitute a new sub-family of RNA binding proteins (Stolow, 1995).
Search PubMed for articles about Drosophila Cabeza
Armstrong, G. A. and Drapeau, P. (2013a). Calcium channel agonists protect against neuromuscular dysfunction in a genetic model of TDP-43 mutation in ALS. J Neurosci 33: 1741-1752. PubMed ID: 23345247
Armstrong, G. A. and Drapeau, P. (2013b). Loss and gain of FUS function impair neuromuscular synaptic transmission in a genetic model of ALS. Hum Mol Genet 22: 4282-4292. PubMed ID: 23771027
Azuma, Y., Tokuda, T., Shimamura, M., Kyotani, A., Sasayama, H., Yoshida, T., Mizuta, I., Mizuno, T., Nakagawa, M., Fujikake, N., Ueyama, M., Nagai, Y. and Yamaguchi, M. (2014). Identification of ter94, Drosophila VCP, as a strong modulator of motor neuron degeneration induced by knockdown of Caz, Drosophila FUS. Hum Mol Genet. PubMed ID: 24497576
Feiguin, F., Godena, V. K., Romano, G., D'Ambrogio, A., Klima, R. and Baralle, F. E. (2009). Depletion of TDP-43 affects Drosophila motoneurons terminal synapsis and locomotive behavior. FEBS Lett 583: 1586-1592. PubMed ID: 19379745
Fujii, R., Okabe, S., Urushido, T., Inoue, K., Yoshimura, A., Tachibana, T., Nishikawa, T., Hicks, G. G. and Takumi, T. (2005). The RNA binding protein TLS is translocated to dendritic spines by mGluR5 activation and regulates spine morphology. Curr Biol 15: 587-593. PubMed ID: 15797031
Immanuel, D., Zinszner, H. and Ron, D. (1995). Association of SARFH (sarcoma-associated RNA-binding fly homolog) with regions of chromatin transcribed by RNA polymerase II. Mol Cell Biol 15: 4562-4571. PubMed ID: 7623847
Jackel, S., Summerer, A. K., Thommes, C. M., Pan, X., Voigt, A., Schulz, J. B., Rasse, T. M., Dormann, D., Haass, C. and Kahle, P. J. (2014). Nuclear import factor transportin and arginine methyltransferase 1 modify FUS neurotoxicity in Drosophila. Neurobiol Dis 74C: 76-88. PubMed ID: 25447237
Kabashi, E., Lin, L., Tradewell, M. L., Dion, P. A., Bercier, V., Bourgouin, P., Rochefort, D., Bel Hadj, S., Durham, H. D., Vande Velde, C., Rouleau, G. A. and Drapeau, P. (2010). Gain and loss of function of ALS-related mutations of TARDBP (TDP-43) cause motor deficits in vivo. Hum Mol Genet 19: 671-683. PubMed ID: 19959528
Kwiatkowski, T. J., Jr., et al. (2009). Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis. Science 323: 1205-1208. PubMed ID: 19251627
Lanson, N. A., Jr., Maltare, A., King, H., Smith, R., Kim, J. H., Taylor, J. P., Lloyd, T. E. and Pandey, U. B. (2011). A Drosophila model of FUS-related neurodegeneration reveals genetic interaction between FUS and TDP-43. Hum Mol Genet 20: 2510-2523. PubMed ID: 21487023
Liu, K. S., Siebert, M., Mertel, S., Knoche, E., Wegener, S., Wichmann, C., Matkovic, T., Muhammad, K., Depner, H., Mettke, C., Buckers, J., Hell, S. W., Muller, M., Davis, G. W., Schmitz, D. and Sigrist, S. J. (2011). RIM-binding protein, a central part of the active zone, is essential for neurotransmitter release. Science 334: 1565-1569. PubMed ID: 22174254
Machamer, J. B., Collins, S. E. and Lloyd, T. E. (2014). The ALS gene FUS regulates synaptic transmission at the Drosophila neuromuscular junction. Hum Mol Genet. PubMed ID: 24569165
Miguel, L., Avequin, T., Delarue, M., Feuillette, S., Frebourg, T., Campion, D. and Lecourtois, M. (2012). Accumulation of insoluble forms of FUS protein correlates with toxicity in Drosophila. Neurobiol Aging 33: 1008 e1001-1015. PubMed ID: 22118902
Polymenidou, M., Lagier-Tourenne, C., Hutt, K. R., Bennett, C. F., Cleveland, D. W. and Yeo, G. W. (2012). Misregulated RNA processing in amyotrophic lateral sclerosis. Brain Res 1462: 3-15. PubMed ID: 22444279
Qiu, H., et al. (2014). ALS-associated mutation FUS-R521C causes DNA damage and RNA splicing defects. J Clin Invest 124: 981-999. PubMed ID: 24509083
Ritson, G. P., Custer, S. K., Freibaum, B. D., Guinto, J. B., Geffel, D., Moore, J., Tang, W., Winton, M. J., Neumann, M., Trojanowski, J. Q., Lee, V. M., Forman, M. S. and Taylor, J. P. (2010). TDP-43 mediates degeneration in a novel Drosophila model of disease caused by mutations in VCP/p97. J Neurosci 30: 7729-7739. PubMed ID: 20519548
Sasayama, H., Shimamura, M., Tokuda, T., Azuma, Y., Yoshida, T., Mizuno, T., Nakagawa, M., Fujikake, N., Nagai, Y. and Yamaguchi, M. (2012). Knockdown of the Drosophila fused in sarcoma (FUS) homologue causes deficient locomotive behavior and shortening of motoneuron terminal branches. PLoS One 7: e39483. PubMed ID: 22724023
Shahidullah, M., Le Marchand, S. J., Fei, H., Zhang, J., Pandey, U. B., Dalva, M. B., Pasinelli, P. and Levitan, I. B. (2013). Defects in Synapse Structure and Function Precede Motor Neuron Degeneration in Drosophila Models of FUS-Related ALS. J Neurosci 33: 19590-19598. PubMed ID: 24336723
Shimamura, M., Kyotani, A., Azuma, Y., Yoshida, H., Binh Nguyen, T., Mizuta, I., Yoshida, T., Mizuno, T., Nakagawa, M., Tokuda, T. and Yamaguchi, M. (2014). Genetic link between Cabeza, a Drosophila homologue of fused in sarcoma (FUS), and the EGFR signaling pathway. Exp Cell Res. PubMed ID: 24928275
Sreedharan, J., Blair, I. P., Tripathi, V. B., Hu, X., Vance, C., Rogelj, B., Ackerley, S., Durnall, J. C., Williams, K. L., Buratti, E., Baralle, F., de Belleroche, J., Mitchell, J. D., Leigh, P. N., Al-Chalabi, A., Miller, C. C., Nicholson, G. and Shaw, C. E. (2008). TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis. Science 319: 1668-1672. PubMed ID: 18309045
Stolow, D. T. and Haynes, S. R. (1995). Cabeza, a Drosophila gene encoding a novel RNA binding protein, shares homology with EWS and TLS, two genes involved in human sarcoma formation. Nucleic Acids Res 23: 835-843. PubMed ID: 7708500
Vance, C., et al. (2009). Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6. Science 323: 1208-1211. PubMed ID: 19251628
Wang, J. W., Brent, J. R., Tomlinson, A., Shneider, N. A. and McCabe, B. D. (2011). The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span. J Clin Invest 121: 4118-4126. PubMed ID: 21881207
Wang, W. Y., Pan, L., Su, S. C., Quinn, E. J., Sasaki, M., Jimenez, J. C., Mackenzie, I. R., Huang, E. J. and Tsai, L. H. (2013). Interaction of FUS and HDAC1 regulates DNA damage response and repair in neurons. Nat Neurosci 16: 1383-1391. PubMed ID: 24036913
Xia, R., Liu, Y., Yang, L., Gal, J., Zhu, H. and Jia, J. (2012). Motor neuron apoptosis and neuromuscular junction perturbation are prominent features in a Drosophila model of Fus-mediated ALS. Mol Neurodegener 7: 10. PubMed ID: 22443542
Zhou, Y., Liu, S., Liu, G., Ozturk, A. and Hicks, G. G. (2013). ALS-associated FUS mutations result in compromised FUS alternative splicing and autoregulation. PLoS Genet 9: e1003895. PubMed ID: 24204307
date revised: 5 April 2015
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